ChIP SimpleChIP Plus Enzymatic Chromatin IP Kit ChIP 2 3 New SimpleChIP Plus Enzymatic Chromatin IP Kit 4 CSTChIP 5 HCT116p53 ChIP 6 7 8 ChIP
クロマチン免疫沈降法 クロマチン免疫沈降 ChIP 法は 細胞のクロマチン中のタンパク質 DNA相互作用を調べる 強力かつ汎用性の高い手法で す 1,2 この手法は 特定のゲノム領域と関係している複数のタンパク質の同定や また逆に ある特定のタンパク質に関係 している多くのゲノム領域の同定に使用されます 3-6 また ChIPアッセイは 特定のタンパク質 DNA相互作用の空間的 経時的関係を明確にするのにも使用されます 例えば 様々なタンパク質因子を遺伝子プロモーターに動員する順序の確定 や遺伝子の活性化における遺伝子座全体の特定のヒストン修飾の相対量を 測定 するのに使用できます 3,4 ヒストンタン パク質の他 転写因子および転写補因子 DNA複製因子 DNA修復タンパク質の結合の解析にも使用可能です ヒストンや非ヒストンタンパク質とDNAを クロスリンクするために 細胞や分離組織を ホルムアルデヒドで固定します ChIPアッセイでは 細胞内のタンパク質 DNA相互作 用を固定 あるいは 保持 する可逆的なタンパク質 DNA架橋剤であるホルムアルデヒドで細胞をまず固定 1,2 します 左図参照 次に 細胞をすすぎ ソニケ 可溶化後 クロマチンを酵素/ ソニケーションで断片化します ーションか酵素消化によりクロマチンを断片化して 特定のタンパク質あるいはヒストン修飾に特異的な抗 ヒストンや非ヒストンタンパク質特異的抗体と Protein G Agarose/Magnetic Beadsを 用いて免疫沈降します 体を用いてクロマチンを免疫沈降します 目的のタン パク質やヒストン修飾に関連するDNA配列は クロス リンクしたクロマチン複合体の一部として共沈降し 脱クロスリンク後 DNAを 分析用に精製します この過程でDNA配列の相対量が増加します 免疫沈降 後にタンパク質 DNAを脱クロスリンクし DNAを精 一般的に ChIPで得られたDNAはスタンダードまたは定量的PCRで分析します その他 ChIPアッセ ゲノムタイリングマイクロアレイ ChIP-on-chip 法 クロー イを シークエンシング ChIP-seq ニング法などと組み合わせ タンパク質 DNA相互作用やヒストン修飾をゲノムレベルで解析すること 5 8 もできます 製します このDNA配列の濃縮物をさまざまな手法で 解析することができます 酵素消化 vs. ソニケーション処理 酵素消化は ソニケーションより穏やかにクロマチンを断片化しますので クロマチンとエピトープを傷つけず 免疫沈降の効 率を向上させます 免疫沈降効率の向上により DNAに結合したタンパク質の検出能が高くなります 標的DNAの検出効率の向上により 試料 時間 試薬の無駄を省けます 酵素消化は 転写因子や補因子と結合したDNAを検出するのに不可欠な 低いバックグラウンドで高い感度を実現します 1 15.8 1 7 5 4 3.7.6 3 PCR primers are specific for known TCF4 binding sites in the CAMK2D and c-myc genes, and a region of the Myt1 gene that does not contain a TCF4 binding site..5.4.3 PCR primers are specific for the transcriptionally active GAPDH and c-myc genes and the inactive Myt1 gene. 25 signal relative to input.9 signal relative to input base pairs 2 15 1.2 5.1 2 1 1 TCF4 (C48H11) Rabbit mab #2569 β-catenin Antibody #9562 Normal Rabbit IgG #2729 Tri-Methyl-Histone H3 (Lys4)(C42D8) Rabbit mab #9751 Acetyl-Histone H3 (Lys9/Lys14) Antibody #9677 Rpb1 CTD (4H8) Mouse mab #2629 2 CAMK2D SimpleChIP CAMK2D Competitor GAPDH SimpleChIP GAPDH Competitor c-myc SimpleChIP c-myc Competitor Myt1 SimpleChIP Myt1 Competitor 酵素消化 Lane 1 とソニケーション法 Lane 2 を採用しているChIPキットを 用いて クロマチンを断片化しました 酵素消化 SimpleChIP したクロマチンを用いると ソニケーション処理 Competitor だけのクロマチンよりも良好な結果が得られます Background References 1. Orlando V (2) Mapping chromosomal proteins in vivo by formaldehyde-crosslinked-chromatin immunoprecipitation. Trends Biochem. Sci. 25(3), 99 14. 2. Kuo MH. et al. (1999) In vivo cross-linking and immunoprecipitation for studying dynamic Protein:DNA associations in a chromatin environment. Methods 19(3), 425 33. 3. Agalioti T. et al. (2) Ordered recruitment of chromatin modifying and general transcription factors to the IFN-beta promoter. Cell 13(4), 667 78. 4. Soutoglou E. et al. (22) Coordination of PIC assembly and chromatin remodeling during differentiation-induced gene activation. Science 295(5561), 191 4. 5. Mikkelsen TS. et al. (27) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448(7153), 553 6. 6. Lee TI. et al. (26) Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125(2), 31 13. 7. Weinmann AS. et al. (22) Identification of unknown target genes of human transcription factors using chromatin immunoprecipitation. Methods 26(1), 37 47. 8. Wells J. et al. (22) Characterizing transcription factor binding sites using formaldehyde crosslinking and immunoprecipitation. Methods 26(1), 48 56. 2 Normal Rabbit IgG #2729 Please visit ZZZ FVWM FR MS for over 14 ChIP validated antibody list
New SimpleChIP Plus Enzymatic Chromatin IP Kits CSTSimpleChIP Plus Enzymatic Chromatin IP DNA CST ChIP ChIP3 G #94 G#95 CST ChIP #95ChIP #96 ChIP-on-chip ChIP-seq #94S SimpleChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads) #95S SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) SimpleChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads) #94: Mouse brain and mouse liver tissue were prepared and disaggregated into a single-cell suspension using a Dounce homogenizer and tissue disaggregator, respectively. The cells were then formaldehyde-crosslinked and chromatin was prepared and digested. DNA was purified and separated by electrophoresis on a 1% agarose gel and stained with ethidium bromide. The majority of chromatin from both brain (lane 1) and liver (lane 2) was digested to 1 to 5 nucleosomes in length (15 to 9 bp). H, M, R, Mk H, M, R, Mk 15 12 1 9 8 7 6 5 4 3 2 1 11, 114, 1 2 SimpleChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads) #94: Mouse brain was prepared and disaggregated into a single-cell suspension using a Dounce homogenizer. The cells were then cross-linked with formaldehyde, and chromatin was prepared and digested. Chromatin immunoprecipitations were performed using the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR using SimpleChIP Mouse GAPDH Intron 2 Primers #8986, SimpleChIP Mouse RPL3 Intron 2 Primers #715, SimpleChIP Mouse HoxA1 Promoter Primers #7341, and SimpleChIP Mouse HoxD1 Intron 1 Primers #7429. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1)..25.2.15.1.5 GAPDH RPL3 HoxA1 HoxD1 Histone H3 (D2B12) XP Rabbit mab (ChIP Formulated) #462 Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mab #9751 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mab #9733 Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mab #9649 Rpb1 CTD (4H8) Mouse mab #2629.12.1.8.6.4.2 GAPDH RPL3 HoxA1 HoxD1 Ezh2 (D2C9) XP Rabbit mab #5246 SUZ12 (D39F6) XP Rabbit mab #3737 RING1B (D22F2) XP Rabbit mab #5694 DNA#95 SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) A SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic beads) #95 B Competitor s Sonication-based Tissue ChIP Kit.4 Histone H3 (D2B12) XP Rabbit mab (ChIP Formulated) #462 Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mab #9751 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mab #9733 Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mab #9649 Rpb1 CTD (4H8) Mouse mab #2629.3 Ezh2 (D2C9) XP Rabbit mab #5246 SUZ12 (D39F6) XP Rabbit mab #3737 RING1B (D22F2) XP Rabbit mab #5694 PCAF (C14G9) Rabbit mab #3378.4 Histone H3 (D2B12) XP Rabbit mab (ChIP Formulated) #462 Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mab #9751 Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mab #9733 Acetyl-Histone H3 (Lys9) (C5B11) Rabbit mab #9649 Rpb1 CTD (4H8) Mouse mab #2629.3 Ezh2 (D2C9) XP Rabbit mab #5246 SUZ12 (D39F6) XP Rabbit mab #3737 RING1B (D22F2) XP Rabbit mab #5694 PCAF (C14G9) Rabbit mab #3378.35.3.25.2.15.1.25.2.15.1.35.3.25.2.15.1.25.2.15.1.5.5.5.5 GAPDH AFM HoxA1 HoxD1 GAPDH AFM HoxA1 HoxD1 GAPDH AFM HoxA1 HoxD1 GAPDH AFM HoxA1 HoxD1 SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic beads) #95 (A) compared to a competitor s sonication-based Tissue Chromatin IP kit (B). Mouse liver was prepared and disaggregated into a single-cell suspension using a tissue disaggregator. The cells were then cross-linked with formaldehyde, and chromatin was prepared and digested according to manufacturer s recommendations. Chromatin immunoprecipitations were performed using the indicated ChIP-validated antibodies. Purified DNA was analyzed by quantitative real-time PCR using SimpleChIP Mouse GAPDH Intron 2 Primers #8986, SimpleChIP Mouse AFM Intron 2 Primers #7269, SimpleChIP Mouse HoxA1 Promoter Primers #7341, and SimpleChIP Mouse HoxD1 Exon 1 Primers #7429. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin (equivalent to 1). 3
CSTChIP #95 SimpleChIP Plus Enzymatic Chromatin IP Kit Magnetic Beads CST #95Histone H3ChIP 1. C57BL/635 mg 2. ChIP CST Mince 15 ml 2 mlbioruptor 1-5 bp1dna15 μg/ml25 μg/ml Histone H3Rabbit IgG RLP3 qpcr2 1. DNA K:, L1 & L2: 2. CST SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) (#95) Histone H3DNA 4 Please visit for over 14 ChIP validated antibody list
HCT116p53 ChIP #95 SimpleChIP Plus Enzymatic Chromatin IP Kit Magnetic Beads CST #95 SimpleChIP Plus Kit PCR 1. p53 p53wt-hct116 p53ko-hct116 8 1 6 Micrococcal Nuclease.5 μl372 2. DNADNA 3. 2.6 μg/chipp53 #2524SHistone H3 Kit Positive ControlNormal Mouse IgG 2 μgchip 4Rotation 14 hr 4. p53p21p53rplnegative Control PCR Relative amount of DNA (Target/Input) Micrococcal Nuclease ChIP Result; PCR n=1 (bp) 1 5 3 2 1 M.5 μl relative amount of DNA (target/input) 2 18 16 14 12 1 8 6 4 2 p53-binding p21 promoter p53ko p53wt α-p53 α-histone lgg relative amount of DNA (target/input) 24 2 18 16 14 12 1 8 6 4 2 RPL3 p53ko p53wt α-p53 α-histone lgg DNA p53ko-hct116 M: Size marker.5 μlmicrococcal Nuclease HCT116 p53wt p53kop21 promoterp53 p53wt p21 promoterp53p53ko p53 p53rpl3p53 p53wt p53kop21 RPL3 Histone H3 Histone Positive ControlNormal Mouse IgG Negative Control p53 SimpleChIP Plus Kitp53 p53ko- HCT116PCRKit in vivoin vitro in vivo ChIPMagnetic Beads 5
DNA 15-9 bp Micrococcal Nuclease 1. CST #95 #94Section I II III125 mg2 1 7 Section III 2. 2. 1 μl 1.5 ml 5 3. Micrococcal Nuclease 3 μl 1 Buffer B + DTT 27 μl1 4. 2. Micrococcal Nuclease μl 2.5 μl 5 μl 7.5 μl 1 μl 37 2 5..5 M EDTA 1 μl 6. 13, rpm 41 7. 1 ChIP Buffer + Protease Inhibitor Cocktail 2 μl 1 8. 3 VirTis Virsonic 1 Ultrasonic Homogenizer/Sonicator 6 1/8-inch probe2 3 HeLaDounce homogenizer 2 9. 1, rpm 41 1. 5 μl 11. 5 μlnuclease-free water 1 μl 5 M NaCl 6 μl RNAse A 2 μl 373 12. RNAse AProteinase K 2 μl652 13. 1 bp DNA2 μl 1%DNA 14. DNA 15-9 bp1-5 DNA Micrococcal NucleaseDNA1 25 mg4 1 6 Micrococcal Nuclease1 Micrococcal Nuclease 5 μl 15-9 bp DNA CSTChIP Section III.5 μl Micrococcal Nuclease 1 15. DNAMicrococcal Nuclease DNA 6 Please visit for over 14 ChIP validated antibody list
SimpleChIP Plus Enzymatic Chromatin IP Kits SimpleChIP Plus Enzymatic Chromatin IPChIPseq #95 SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) ChIP-seq #95G #96 salmon sperm DNADNA CST ChIP Micrococcal Nuclease CST 14 1 6 25 mg Micrococcal Nuclease15-9 bp 4 1 6 25 mg Micrococcal Nuclease.5 μl Micrococcal Nuclease SimpleChIP Plus Enzymatic Chromatin IPBuffer A Buffer B Micrococcal Nuclease SimpleChIP Plus Enzymatic Chromatin IP IV. CST IP 1% 1 DNA2DNA DNA-- --- 15-9 bp DNA OD 26 125-25 μg/ml 15bp Appendix B *15 bp 6 1bp 7bp 5bp 4bp 3bp 2bp 1bp 1 2 IP CST IP14 1 6 25 mg 1-2 μg IP 1 1 6 2.5-5 μgipip 5 μl1 ChIP Buffer CST IP 25 mg HeLa 4 1 6 DNA Medimachine (BD Biosciences) Dounce Homogenizer Medimachine Dounce HomogenizerIP Medimachine Dounce Homogenizer ChIP 5-1 μg / DNA 25 mg 2-3 μg 2-3 μg/ml 25 mg 1-15 μg 1-15 μg/ml 25 mg 8-1 μg 8-1 μg/ml 25 mg 2-5 μg 2-5 μg/ml 25 mg 2-5 μg 2-5 μg/ml HeLa 4 1 6 1-15 μg 1-15 μg/ml ChIP CST ChIP IP SimpleChIP Plus Enzymatic Chromatin IP Recommended Antibody Dilutions ChIP CST4 1 6 1-2 μg CST ChIPChIP ChIP IP ChIP1 2-5 μg SimpleChIP Plus Enzymatic Chromatin IPG #94 G#95 IP #717 6-Tube Magnetic Separation Rack DNA ChIP-seq salmon sperm DNA DNA 7
New SimpleChIP Plus Enzymatic Chromatin IP Kits ChIP ( ) #94S SimpleChIP Plus Enzymatic Chromatin IP Kit (Agarose Beads) 11, #95S SimpleChIP Plus Enzymatic Chromatin IP Kit (Magnetic Beads) 114, Over 14 ChIP Validated Antibodies CSTChIP ChIP www.cstj.co.jp/technologies/chip.php SimpleChIP Control PCR Primers 2 ChIP DNA ChIP DNA SimpleChIP Assay Kits ChIPPCR ( ) #898S SimpleChIP Stem Cell Master Regulator Assay Kit 126, #8982S SimpleChIP Human Bivalent Promoter Assay Kit 15, #8981S SimpleChIP Mouse Bivalent Promoter Assay Kit 15, ChIP 212 11 212 Cell Signaling Technology, Inc. XP, SimpleChIP, and Cell Signaling Technology are trademarks of Cell Signaling Technology, Inc. Antibodies and Related Reagents for Signal Transduction Research